1. Field of the Invention
The present invention relates to polypeptides having aninopeptidase activity and isolated nucleic acid sequences encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the nucleic acid sequences as well as methods for producing the polypeptides. The present invention further relates to methods of obtaining protein hydrolysates useful as flavour improving agents.
2. Description of the Related Art
Various food products, e.g., soups, sauces and seasonings, contain flavoring agents obtained by hydrolysis of proteinaceous materials. This hydrolysis is conventionally accomplished using strong hydrochloric acid, followed by neutralization with sodium hydroxide. However, such chemical hydrolysis leads to severe degradation of the amino acids obtained during the hydrolysis, and also to hazardous byproducts formed in the course of this chemical reaction. Increasing concern over the use of flavoring agents obtained by chemical hydrolysis has led to the development of enzymatic hydrolysis processes.
Enzymatic hydrolysis processes of proteinaceous materials aim at obtaining a high degree of hydrolysis (DH), and this is usually attained using a complex of unspecific acting proteolytic enzymes (i.e., unspecific-acting endo- and exo-peptidases). For example, WO 94/25580 describes a method for hydrolyzing proteins by use of an unspecific acting enzyme preparation obtained from Aspergillus oryzae. Specific acting proteolytic enzymes have not been used for this purpose because such enzymes only lead to an inadequate degree of hydrolysis.
Polypeptides having aminopeptidase activity catalyze the removal of one or more amino acid residues from the N-terminus of peptides, polypeptides, and proteins. Such polypeptides are classified under the Enzyme Classification Number E.C. 3.4.11.- of the International Union of Biochemistry and Molecular Biology.
WO 96/28542 discloses an aminopeptidase which has a moleculer weight of 35 kDa. JP-7-5034631 (Noda) discloses a leucine aminopeptidase obtained from yellow koji mold, which includes Aspergillus oryzae. JP-7-4021798 (Zaidan Hojin Noda Sangyo) discloses the production of miso by adding a leucine aminopeptidase II prepared by cultivating a number of strains, including Aspergillus oryzae strain 460 (ATCC 20386) and strain IAM 2616. Aspergillus oryzae strain 460 is known to produce a number of leucine aminopeptidases of which three have a molecular weight of 26.5, 56, and 61 kDa by gel filtration (Nakada et al., 1972, Agricultural and Biological Chemistry 37: 757-765; Nakada et al., 1972, Agricultural and Biological Chemistry 37: 767-774; and Nakada et al., 1972, Agricultural and Biological Chemistry 37: 775-782; respectively). Penicillium citrium produces an intracellular leucine aminopeptidase with a molecular weight of 65 kDa by SDS-PAGE (Kwon et al., 1996, Journal of Industrial Microbiology 17: 30-35). 
WO 97/04108 (Roehm) discloses DNA encoding an Aspergillus sojaeleucine aminopeptidase. Chang and Smith (1989, Journal of Biological Chemistry 264: 6979-6983) disclose the molecular cloning and sequencing of a gene encoding a vacuolar aminopeptidase from Saccharomyces cerevisiae. Chang et al. (1992, Journal of Biological Chemistry 267: 8007-8011) disclose the molecular cloning and sequencing of a gene encoding a methionine aminopeptidase from Saccharomyces cerevisiae. 
So The production of protein hydrolysates with desirable organoleptic properties and high degrees of hydrolysis generally requires the use of a mixture of peptidase activities. It would be desirable to provide a single component peptidase enzyme which has activity useful for improving the organoleptic properties and degree of hydrolysis of protein hydrolysates used in food products either alone or in combination with other enzymes.
It is an object of the present invention to provide improved polypeptides having aminopeptidase activity as well as methods for obtaining protein hydrolysates with desirable organoleptic qualities and high degrees of hydrolysis.
The present invention relates to isolated polypeptides having aminopeptidase activity selected from the group consisting of:
(a) a polypeptide having an amino acid sequence which has at least 50% identity with the amino acid sequence of SEQ ID NO:2;
(b) a polypeptide encoded by a nucleic acid sequence which hybridizes under medium stringency conditions with (i) the nucleic acid sequence of SEQ ID NO:1, (ii) its complementary strand, or (iii) a subsequence thereof;
(c) an allelic variant of (a) or (b); and
(d) a fragment of (a), (b), or (c), wherein the fragment has aminopeptidase activity; and
(e) a polypeptide having aminopeptidase activity with physicochemical properties of (i) a pH optimum in the range of from about pH 7.27 to about pH 10.95 determined at ambient temperature in the presence of Ala-para-nitroanilide; (ii) a temperature stability of 90% or more, relative to initial activity, at pH 7.5 determined after incubation for 20 minutes at 60xc2x0 C. in the absence of substrate; and (iii) an activity towards Xaa-para-nitroanilide wherein Xaa is selected from the group consisting of Leu, Glu, Gly, Ala, and Pro.
The present invention also relates to isolated nucleic acid sequences encoding the polypeptides and to nucleic acid constructs, vectors, and host cells comprising the nucleic acid sequences as well as methods for producing the polypeptides.
The present invention also relates to methods for obtaining hydrolysates from proteinaceous substrates which comprise subjecting the proteinaceous material to a polypeptide with aminopeptidase activity alone or in combination with an endopeptidase, and to hydrolysates obtained from the method.
The present invention also relates to methods for obtaining from a proteinaceous substrate a hydrolysate enriched in free glutamic acid and/or peptide bound glutamic acid residues, which methods comprise subjecting the substrate to a deamidation process and to the action of a polypeptide having aminopeptidase activity.
The present invention further relates to flavor-improving compositions comprising a polypeptide with aminopeptidase activity. The compositions may further comprise additional enzymatic activities.
In a final aspect, the methods of the invention may be used in food related applications to improve flavor, such as baking. Alternatively, flavor improvement in foods may be achieved by the addition of hydrolysates obtained by the methods of the invention.